CRISPR/Cas9-engineering of HMC-1.2 cells renders a human mast cell line with a single D816V-KIT mutation: An improved preclinical model for research on mastocytosis.

The HMC-1.2 human mast cell (huMC) line is often employed in the study of attributes of neoplastic huMCs as found in patients with mastocytosis and their sensitivity to interventional drugs in vitro and in vivo. HMC-1.2 cells express constitutively active KIT, an essential growth factor receptor for huMC survival and function, due to the presence of two oncogenic mutations (D816V and V560G). However, systemic mastocytosis is commonly associated with a single D816V-KIT mutation. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9-engineering to reverse the V560G mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single mono-allelic D816V-KIT variant. Transcriptome analyses predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia in HMC-1.3 compared to HMC-1.2 cells, with differences in expression of molecular components and cell surface markers. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. However, in liquid culture conditions, the growth of HMC-1.2 and HMC-1.3 cells was comparable. Phosphorylation levels of ERK1/2, AKT and STAT5, representing pathways associated with constitutive oncogenic KIT signaling, were also similar between HMC-1.2 and HMC-1.3 cells. Despite these similarities in liquid culture, survival of HMC-1.3 cells was diminished in response to various pharmacological inhibitors, including tyrosine kinase inhibitors used clinically for treatment of advanced systemic mastocytosis, and JAK2 and BCL2 inhibitors, making HMC-1.3 more susceptible to these drugs than HMC-1.2 cells. Our study thus reveals that the additional V560G-KIT oncogenic variant in HMC-1.2 cells modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases the tumorigenicity, suggesting that engineered huMCs with a single D816V-KIT variant may represent an improved preclinical model for mastocytosis.

Emerging mechanisms contributing to mast cell-mediated pathophysiology with therapeutic implications.

Mast cells are tissue-resident immune cells that play key roles in the initiation and perpetuation of allergic inflammation, usually through IgE-mediated mechanisms. Mast cells are, however, evolutionary ancient immune cells that can be traced back to urochordates and before the emergence of IgE antibodies, suggesting their involvement in antibody-independent biological functions, many of which are still being characterized. Herein, we summarize recent advances in understanding the roles of mast cells in health and disease, partly through the study of emerging non-IgE receptors such as the Mas-related G protein-coupled receptor X2, implicated in pseudo-allergic reactions as well as in innate defense and neuronal sensing; the mechano-sensing adhesion G protein-coupled receptor E2, variants of which are associated with familial vibratory urticaria; and purinergic receptors, which orchestrate tissue damage responses similarly to the IL-33 receptor. Recent evidence also points toward novel mechanisms that contribute to mast cell-mediated pathophysiology. Thus, in addition to releasing preformed mediators contained in granules and synthesizing mediators de novo, mast cells also secrete extracellular vesicles, which convey biological functions. Understanding their release, composition and uptake within a variety of clinical conditions will contribute to the understanding of disease specific pathology and likely lead the way to novel therapeutic approaches. We also discuss recent advances in the development of therapies targeting mast cell activity, including the ligation of inhibitory ITIM-containing receptors, and other strategies that suppress mast cells or responses to mediators for the management of mast cell-related diseases.

The DNA methyltransferase inhibitor, guadecitabine, targets tumor-induced myelopoiesis and recovers T cell activity to slow tumor growth in combination with adoptive immunotherapy in a mouse model of breast cancer.

BACKGROUND: Myeloid derived suppressor cells (MDSCs) present a significant obstacle to cancer immunotherapy because they dampen anti-tumor cytotoxic T cell responses. Previous groups, including our own, have reported on the myelo-depletive effects of certain chemotherapy agents. We have shown previously that decitabine increased tumor cell Class I and tumor antigen expression, increased ability of tumor cells to stimulate T lymphocytes, depleted tumor-induced MDSC in vivo and augmented immunotherapy of a murine mammary carcinoma. RESULTS: In this study, we expand upon this observation by testing a next-generation DNA methyltransferase inhibitor (DNMTi), guadecitabine, which has increased stability in the circulation. Using the 4 T1 murine mammary carcinoma model, in BALB/cJ female mice, we found that guadecitabine significantly reduces tumor burden in a T cell-dependent manner by preventing excessive myeloid proliferation and systemic accumulation of MDSC. The remaining MDSC were shifted to an antigen-presenting phenotype. Building upon our previous publication, we show that guadecitabine enhances the therapeutic effect of adoptively transferred antigen-experienced lymphocytes to diminish tumor growth and improve overall survival. We also show guadecitabine's versatility with similar tumor reduction and augmentation of immunotherapy in the C57BL/6 J E0771 murine breast cancer model. CONCLUSIONS: Guadecitabine depleted and altered MDSC, inhibited growth of two different murine mammary carcinomas in vivo, and augmented immunotherapeutic efficacy. Based on these findings, we believe the immune-modulatory effects of guadecitabine can help rescue anti-tumor immune response and contribute to the overall effectiveness of current cancer immunotherapies.

A new look at IgE beyond allergies.

Immunoglobulin E (IgE), though constitutively present at low levels, is most commonly studied in atopic disease where it plays a vital role in mast cell degranulation and in initiating a T helper 2 (Th2) response. With the advent of better detection assays, however, researchers are discovering the importance of IgE in actively contributing to many disease states and pathologies. This review will discuss the latest findings in IgE beyond its role in allergies and recently discovered roles for IgE in its cell-bound form on FcεRI-expressing effector cells like monocytes and dendritic cells. In terms of parasites, we will discuss helminth-induced IgE that appears to protect the worms from immune recognition and a tick-borne illness that elicits an IgE response against red meat. Next, we describe recent findings of how auto-reactive IgE can contribute to the progression of lupus and induce organ damage. Finally, we summarize the emerging roles of IgE in tumor surveillance and antibody-dependent cytotoxicity. We additionally discuss recent or ongoing clinical trials that either target harmful IgE or use the unique characteristics of the isotype.

B1 Cell IgE Impedes Mast Cell-Mediated Enhancement of Parasite Expulsion through B2 IgE Blockade.

Helminth infection is known for generating large amounts of poly-specific IgE. Here we demonstrate that innate-like B1 cells are responsible for this IgE production during infection with the nematode parasites Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. In vitro analysis of B1 cell immunoglobulin class switch recombination to IgE demonstrated a requirement for anti-CD40 and IL-4 that was further enhanced when IL-5 was added or when the B1 source was helminth infected mice. An IL-25-induced upregulation of IgE in B1 cells was also demonstrated. In T cell-reconstituted RAG1-/- mice, N. brasiliensis clearance was enhanced with the addition of B2 cells in an IgE-dependent manner. This enhanced clearance was impeded by reconstitution with IgE sufficient B1 cells. Mucosal mast cells mediated the B2 cell enhancement of clearance in the absence of B1 cells. The data support B1 cell IgE secretion as a regulatory response exploited by the helminth.

ADAM10-Mediated ICOS Ligand Shedding on B Cells Is Necessary for Proper T Cell ICOS Regulation and T Follicular Helper Responses.

The proper regulation of ICOS and ICOS ligand (ICOSL) has been shown to be essential for maintaining proper immune homeostasis. Loss of either protein results in defective humoral immunity, and overexpression of ICOS results in aberrant Ab production resembling lupus. How ICOSL is regulated in response to ICOS interaction is still unclear. We demonstrate that a disintegrin and metalloproteinase (ADAM)10 is the primary physiological sheddase of ICOSL in mice and humans. Using an in vivo system in which ADAM10 is deleted only on B cells, elevated levels of ICOSL were seen. This increase is also seen when ADAM10 is deleted from human B cell lines. Identification of the primary sheddase has allowed the characterization of a novel mechanism of ICOS regulation. In wild-type mice, interaction of ICOS/ICOSL results in ADAM10-induced shedding of ICOSL on B cells and moderate ICOS internalization on T cells. When this shedding is blocked, excessive ICOS internalization occurs. This results in severe defects in T follicular helper development and TH2 polarization, as seen in a house dust mite exposure model. In addition, enhanced TH1 and TH17 immune responses are seen in experimental autoimmune encephalomyelitis. Blockade of ICOSL rescues T cell ICOS surface expression and rescues, at least in part, T follicular helper numbers and the abnormal Ab production previously reported in these mice. Overall, we propose a novel regulation of the ICOS/ICOSL axis, with ADAM10 playing a direct role in regulating ICOSL, as well as indirectly regulating ICOS, thus controlling ICOS/ICOSL-dependent responses.